PROJECT SUMMARY The broad objective of this research proposal is to advance our understanding of thyroid physiology through the study of genetic defects at key regulatory processes. In addition to identification of new syndromes and defects in structural genes, research centers on regulation of gene expression and epigenetic effects. The program owes its success to the worldwide referral of patient material, to the clinical and technical skills of the PI's laboratory, and to collaborative arrangements with accomplished investigators from the US and abroad that provide complementary knowledge and technical expertise. The proposal encompasses three aims. (1) Discovery of new genes and regulators of gene expression responsible for congenital and inherited thyroid diseases. Material will be derived from the PI's collection of 4,647 serum and DNA samples from 1,161 families, of which the genetic defect was identified in only 783 families using the candidate gene approach. The key cases of the remaining 418 families will be submitted to targeted next-generation sequencing (NGS). Those that fail to show known thyroid gene defects (approximately 250 familes, based on a pilot study) will be submitted first to whole exome sequencing and a subgroup without exonic mutations will undergo further whole genome sequencing. It is anticipated that confirmation of mutations by Sanger sequencing and cosegregation studies, will lead to the discovery of new genes and/or mechanisms of congenital thyroid disorders. These will be characterized in greater detail with functional studies and in transgenic mouse models. (2) Acquired resistance to thyroid hormone (RTH) due to fetal exposure to high thyroid hormone (TH) levels that persists into adulthood will be studied in humans and in mice. In humans, transmission of this putative epigenetic effect to the next generation (F2) will be studied. In a mouse model, the precise mechanism of this effect will be determined through biochemical and genetic studies in tissues. Whether RTH develops in humans after prolonged exposure to high levels of TH during adulthood, will also be determined. (3) The PI recently identified mutations in a short tandem repeat (STR) in a region of chromosome-15 that results in a dominantly inherited form of resistance to thyrotropin (RTSH). These mutations manifest with increased expression of two thyroid specific micro RNAs, as well as an EST (AV707477) downstream from the STR. This region on chromosome-15 is unique to humans and higher primates. The latter precludes the use of a mouse model for further studies. Thus, a functional thyroid cell line needs to be identified to allow for probing into the mechanism of this RTSH form using site- directed mutagenesis. Several alternatives research plans are proposed one of which is the generation of functional WT and mutant thyroid cell organoids. The latter will be produced from WT and the patients' induced pluripotent stem cells, derived from their skin fibroblasts. The proposed research will result in the discovery of new genes and mechanisms causing congenital and inherited thyroid diseases. In addition to knowledge gained regarding thyroid physiology and pathophysiology, these studies will provide the means for rapid and specific diagnosis and for rational treatment.